[ACC] Product Overview

Sejak 1974, ACC jadi pelopor reagen LAL dan deteksi glukan. Pemegang lisensi FDA pertama, ACC hadir sebagai standar emas dalam pengujian endotoksin di dunia laboratorium.

Associates of Cape Cod, Inc. - a Seikagaku Group Company www.acciusa.com • 508.540.3444 Associates of Cape Cod, Inc. Your Endotoxin & Glucan Experts

BEING FIRST MEANS DOING SOMETHING NO ONE ELSE HAS EVER DONE BEFORE... WE DO THAT A LOT.

1st To Introduce An Animal Free, Recombinant LAL Reagent – PyroSmart NextGen ®

1st Large Scale IVF Program To Introduce Horseshoe Crabs Into The Wild

1st To Establish BET Contract Testing Services

1st BET Company Licensed By The FDA

Products & Services Catalog

Advance your laboratory’s Endotoxin and Glucan detection capabilities into 1 st place with Associates of Cape Cod, Inc. today!

ISO 13485:2016 DEVICES NSAI Certified MEDICAL

YOUR ENDOTOXIN SENSITIVITY, FLEXIBILITY AND COMPLIANCE

Contents Your Goals - Your Endotoxin Experts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ................................. 4 Attainable Sustainability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...................................... 5 Horseshoe Crabs And The Biomedical Industry... Know The Truth .. . . . . . . . . . . . . . . . . . . ..................... 6-7 BET Methodology and Support Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ................................ 9 BET Method Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..................................... 10 Methodology & Applications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .................................... 10 Kinetic Testing Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .................................... 10 Bacterial Endotoxin Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..................................... 11 Gel-Clot BET Testing Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .................................. 11 Overview of Testing Procedures. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .................................. 11 Support Services. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....................................... 12 BEST QC Microbiology Training Program. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .............................. 14 Kinetic Endotoxin Detection Systems .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ................................ 15 PyroSmart NextGen ® Recombinant Cascade Reagent (rCR) . . . . . . . . . . . . . . . . . . . . . . ....................... 16-19 Pyrochrome ® Reagent Chromogenic Endotoxin Testing. . . . . . . . . . . . . . . . . . . . . . . . . ......................... 20 Chromo-LAL Kinetic Chromogenic Endotoxin Testing . . . . . . . . . . . . . . . . . . . . . . . . . . ......................... 21 Pyrotell ® -T Reagent Turbidimetric Endotoxin Testing . . . . . . . . . . . . . . . . . . . . . . . . . . .......................... 22 Pyros Kinetix ® Flex Incubating Kinetic Tube Reader. . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............................ 23 ELx808 IU™ Absorbance 96 Well Microplate Reader. . . . . . . . . . . . . . . . . . . . . . . . . . .......................... 24 Pyros ® eXpress Endotoxin and Glucan Analysis Software. . . . . . . . . . . . . . . . . . . . . . . . . ........................ 25 Kinetic System Summary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .................................... 26 Gel-Clot Endotoxin Detection Testing .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ................................ 27 Pyrotell ® Gel-clot Endotoxin Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ................................ 28 Pyrosate ® Kit Rapid Endotoxin Detection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .............................. 29 Gel-Clot Testing Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ................................... 30-31 Glucan Detection .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........................................ 33 Glucatell ® Kit (1 g 3)- b - D -Glucan Detection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............................. 34 Glucan Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....................................... 35 BET Accessory Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...................................... 37 Pyroclear ® Disposable Products. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .................................. 38 LAL Reagent Water (LRW) Disposable Products. . . . . . . . . . . . . . . . . . . . . . . . . . . . ............................ 38 Pyrotubes ® Disposable Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ................................. 38 Pyroplates ® Disposable Products. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ................................. 38 Precision Pipette Tips Disposable Products. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .............................. 38 Pyrosol ® LAL Reconstitution Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ................................ 38 Glucashield ® (1 g 3)- b - D -Glucan Inhibiting Buffer. . . . . . . . . . . . . . . . . . . . . . . . . . . . ........................... 38 Control Standard Endotoxin (CSE). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ................................. 39 Contract Test Service . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....................................... 40 CTS Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....................................... 42 Drug & Medical Device Testing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .................................. 44 Custom Services. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....................................... 45 Contact Information. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...................................... 45 Services Offered. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....................................... 46 Ordering Information and Sales Offices .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............................... 47

EXPERTS IN BACTERIAL ENDOTOXIN TESTING

We Are ACC... Your Endotoxin Experts

Here at Associates of Cape Cod, Inc., (ACC), we work hard to offer you the highest quality endotoxin testing reagents, disposables and systems available. No matter where your company is located, we offer on-site expertise and local support for all your bacterial endotoxin testing needs. With nearly 50 years in this industry, we’ve established ourselves as: • Outstanding BET industry Customer and Technical Support • Operating at the Highest Quality Standards in our industry (certified to ISO 13485:2016) • Celebrating nearly 50 years as the first to commercially manufacture LAL • Specializing in Validation Process and Services • The only “pure player” endotoxin detection company in the world • Offering the most sensitive reagents in the industry

YOUR GOALS YOUR ENDOTOXIN EXPERTS

A Partnership In Change Where do you start? Bacterial Endotoxin Testing is an essential tool for quality control of raw materials, screening of in-process samples and maintaining your in-process water system as well as a regulatory required test to release your finished products. Whether you’re bringing testing in house, looking to switch from gel-clot to kinetic, would like to validate a sustainable recombinant reagent, or want to convert to ACC as your supplier, we’ve done it all. With nearly 50 years of experience in the BET industry, we have your answers.

GOAL! Successful Validated Change

7 – Implement new processes in QMS

6 – Train Staff

5 – Write Procedures

4 – Validate Samples

While ACC’s approach will be tailored specifically for your company and your goals, a basic breakdown of this process is outlined here.

3 – Validate Systems/Equipment

2 – Method Development of Samples

1 – Qualify ACC as Supplier

ATTAINABLE SUSTAINABILITY

ACC’s Horseshoe Crab Sustainability Project

Internally Funded Initiative

now and for future generations to come. Working with local regulators and having received a class 1 type 4 aquaculture permit and utilizing a patent pending process, ACC collects HSC eggs, fertilizes, grows and strategically releases horseshoe crabs back into their natural environment. This program only utilizes eggs collected from bait crabs that are sacrificed for the eel, conch and whelk fisheries, extending their genetic legacy for generations to come. Please visit www.acciusa.com for more information and future updates!

Associates of Cape Cod, Inc.’s One Of A Kind Sustainability Project Meets A Milestone Of Over One Million Juvenile Crabs Released In Massachusetts! Associates of Cape Cod, Inc. (ACC) introduced our Horseshoe Crab Sustainability Project in 2018. The system worked so well, in 2019 systems were allocated as grants to organizations in an effort to help horseshoe crabs in Asia. To date the project has reared and released into coastal waters, more than 1 million juvenile horseshoe crabs! This unique program was aimed at complementing our over nearly 50 year history of horseshoe crab conserva- tion and to ensure a stable supply of horseshoe crabs

ATTAINABLE HORSESHOE CRABS AND THE BIOMED

What makes a horseshoe crabs’ blood so special? Horseshoe crab blood carries factors that react to antigens found on and in gram negative bacteria walls by forming a clot around it. The clot isolates the bacteria, and protects the crab from infection. The blood also begins a healing process similar to ours where we form a clot, a scab, and eventually wounds heal. What makes LAL so important? The LAL test is the most sensitive, accurate and cost effective test on the market today to detect contaminating endotoxins. This test was first licensed by the FDA in the 1970’s, and is now the gold standard. It can detect endotoxin in the parts per billion. That’s like finding a grain of sand in an Olympic swimming pool. Prior to LAL, rabbits were used to test for endotoxin by injecting the rabbit with samples of the product being manufactured and waiting two or three days to see if the rabbit developed a fever. Hundreds of thousands of rab- bits were required to be held and utilized this way. LAL based assays replace this test with one that is more humane, more accurate, cost effective and can give results in a test tube, in about an hour. There are very few people you are likely to meet in your lifetime who have not benefited from a bacterial endotoxins test. What types of things are tested with the blood? The FDA has mandated (it is the law) that all injectable or indwelling materials must be tested for endotoxin contamination before being released for sale. This is to protect the public from products that are not sufficiently free of materials that can make a patient ill from exposure to gram-negative cell wall material. If endotoxin enters your blood stream it can make you sick and possibly even kill you. So the test we manufacture is used for medical devices, such as knee replacements, stents, heart valves, intravenous solutions; and drugs and vaccines like childhood immunizations, insulin, flu vaccine and chemotherapy drugs to name a few. Anything injected or implanted into the human body must be free of endotoxin.

I have read somewhere crab blood is worth $15,000 a quart. Is this true?

Absolutely not, this is a myth sensationalized by some me- dia. Manufacturing LAL which is made from the white blood cells of horseshoe crabs is a complex process that is regulated by the FDA and must be done under extremely clean condi- tions. A typical LAL test costs less than $20. In terms of the impact it has

had on human health and safety, it is safe to say it has saved many lives and is therefore priceless. Where do the crabs you bleed come from?

Most of the crabs that come to our facility are from Massachu- setts waters, Vineyard Sound, Nantucket Sound, and Buzzards Bay. Fisherman catch them a number of different ways but must follow strict regulations on size, number of crabs har- vested, and quota management.

How does the process of bleeding crabs work? The process is very similar to when people donate blood. The crabs are checked for good health, placed in a very clean laboratory, where we disin- fect a portion of the shell, and carefully insert a sterile needle. The crabs have a sinus in the dorsal aspect of their body just under the shell that holds excess blood, we collect from that region. The way the crabs are held, limits the blood that can be harvested to the dorsal sinus, the majority of the blood which is in the gill area is untouched. Studies have shown that the crabs tolerate this process very well and the overwhelming majority survives. What threats face the horseshoe crabs today, are they endangered? Like any sea creature, horseshoe crabs are dependent on a suitable environment in which to live and reproduce. Water quality is an important

SUSTAINABILITY ICAL INDUSTRY... KNOW THE TRUTH

factor as is having suitable beaches in which to lay their eggs. Fertilizers, septic systems, and other forms of pollution can greatly reduce the quality of water on which the crabs depend. Sea walls, rip-rap and jetty’s can manipulate the natural movement of sand on beaches and affect spawning habitat. Beach nourishment, the practice of bringing in truckloads of sand to beaches to replenish what’s lost, or make them look nice, can bury millions of eggs before they hatch if not carefully timed. Crabs are also used as bait for conch and eels which is another source of man-made mortality.

Best Management Practices (BMPs) for the industry. We also collect data for the regulators from every crab that enters our facility, which is invaluable to understanding population dynamics. Most recently ACC has implemented a one of a kind sustainability project where we can create juvenile crabs in-vitro and release them to the wild. You can learn more about this exciting new program here. What information should more people know about horseshoe crabs? Horseshoe crabs and their ancestors have been on this planet for somewhere around 400 million years, they have survived mass extinctions. They are not harmful, don’t sting, bite or try to do us any harm. Remember when you see a horseshoe crab shell washed up on the beach it is likely a molt, and not a dead crab. Crabs can only grow by shedding their shells and growing larger ones. Old shells are discarded and many beachcombers worry crabs are dying when they are really just growing up. Even as recent as the 1950s crabs were destroyed by the tens of thousands by people on Cape Cod and elsewhere fearing they were harmful to shellfish beds or for use as fertilizer and pig food. In fact, they are useful for shell fisherman by helping to till and keep sediment aerated. They are an important part of the international ecosystem. What can I do? Water quality and human development are major threats to all fragile ecosystems such as the embay- ments where horseshoe crabs reproduce and grow. Do your part in limiting the impact humans have on water quality and beach erosion. If you ever see a crab upside down on the beach, gently roll it over so it can return to the water. And remember; the next

Crabs in the United States are regu- lated and monitored carefully. They are not endangered, in fact, in many areas populations are grow- ing considerably. In other parts of the world, they are victims of pollution and humankind’s de-

velopment of coastal areas and are not so closely monitored.

What does ACC do to support conservation? ACC has always promoted and practiced a catch and release fishery where the overwhelming majority of crabs survive the process of blood extraction. We work closely with fisherman and regulators to mini- mize the impact we may have on crab populations. ACC was instrumental in creating a minimum size limit for crabs to ensure only mature crabs are collected, and helping to keep a biomedical only fishery in Pleasant Bay MA where all the crabs collected are released. We have supported con- servation efforts that include the use of bait bags, decreased catch limits and prohibition of fishing for crabs around peak spawning periods. We also participate in the Massachusetts “rent a crab program” where crabs destined for use as bait are brought to our facility first. This helps to limit the overall impact on crabs, and is unique to Massachusetts. ACC takes part in the Atlantic States Marine Fisheries Commission (ASMFC) Horseshoe Crab Advisory Panel where we helped develop the

time you or a loved one receives an injection, IV or implant, be sure to thank a horseshoe crab!

THROUGH FREE CONSULTATION AND SUPPORT WE WILL ASSIST YOU TO DETERMINE THE METHOD THAT BEST SUITS YOUR NEEDS AND SAMPLE TYPE. ACC OFFERS ALL REGULATORY COMPLIANT KINETIC AND GEL-CLOT BET METHODS.

BET METHODOLOGY AND

SUPPORT SERVICES

Our Test, Your Cure...

Ensuring A Healthy World

Your Endotoxin Experts! With the best training and technical support in the field, we’ll partner with you every step of the way.

BET METHOD OPTIONS

Methodology & Applications Through free consultation and support we will assist you to determine the method that best suits your needs and sample type. ACC offers all regulatory compliant kinetic and gel-clot BET methods. Introduction Limulus Amebocyte Lysate (LAL) tests detect and quantify bacterial endotoxins derived from the outer cell wall membrane of gram-negative bacteria. The critical component of the LAL reagents used in endo- toxin tests is derived from blood cells (amebocytes) of the horseshoe crab, Limulus polyphemus. Amebocytes contain the proteins of the blood clotting mechanism, which is triggered primarily by endotoxins and also by (1 g 3)- b - D -Glucan. LAL reagents are primarily used to test for endotoxins in injectable pharmaceuticals, bi- ological products, medical devices and renal dialysis centers. Endotoxin tests are described in the Bacterial Endotoxins Test chapter in the United States Pharma- copeia (Chapter <85>) and in the equivalent chapters in the European Pharmacopoeia (Chapter 2.6.14) and the Japanese Pharmacopoeia (General Tests, No. 4.01). Modified LAL reagents can be used for specific detec- tion of (1 g 3)- b - D -Glucans. Selecting a Method Consider the following when deciding which Bacterial Endotoxins Test method to use:

endotoxin concentrations and greater dilution of sam- ples, which is important for overcoming interference. Both kinetic methods utilize software to quantify your test results. The gel-clot method is a simple, positive/ negative, low start-up cost alternative that has been the reference method for years. Kinetic Testing Methods Chromogenic Method The BET reagent is formulated with a synthetic sub- strate which produces a chromophore when cleaved by endotoxin activated enzymes. • Requires either the Pyros Kinetix ® Flex tube reader or an incubating plate reader system such as the BioTek ELX808 IU TM * • Maximum sensitivity to 0.001 EU/mL, highest chro- mogenic sensitivity available in the BET industry when using ACC’s Pyrochrome ® reagent

• Electronically stored data

• Incubation time varies depending on the standard curve range • High sensitivity allows for greater dilution to over- come interference Turbidimetric Method The optical density (turbidity) increase that accompa- nies the clotting reaction is read in our Pyros Kinetix ® Flex tube reader or in an incubating microplate reader. • Requires either the Pyros Kinetix ® Flex tube reader system or an incubating microplate reader such as the BioTek ELX808 IU TM * • Maximum sensitivity to 0.001 EU/mL, highest sensi- tivity available in the BET industry when using ACC’s Pyrotell ® -T reagent • Quantitative test results and electronically stored data • Incubation time varies depending on the standard curve range. Results can be obtained in as little as 15 minutes with ACC reagents

• What are the regulatory requirements, if any?

• What type of product or sample is to be tested?

• What test sensitivity is required? (What is the endotoxin limit specification for the sample?)

• Is quantitative analysis desired?

There are three principal Bacterial Endotoxins Test methods: the chromogenic, turbidimetric and gel-clot methods. The first two may be grouped together as kinetic photometric methods as they require a timed optical reader. Both chromogenic and turbidimetric methods offer the greatest sensitivity, allowing detection of low

*Trademark of BioTek Instruments, Inc.

BACTERIAL ENDOTOXIN TESTING

• High sensitivity allows for greater dilution to over- come interference Gel-Clot BET Testing Method Gel-clot Method The formation of a gel-clot indicates the presence of endotoxin in a sample. The method is performed in small test tubes and is read manually by inverting the test tubes. • Requires non-circulating water bath or dry bath incubator

• Manually read test

or potential interference. It is typically performed by testing a series of dilutions of sample with and with- out a Positive Product Control (PPC). PPCs consist of sample with a known amount of endotoxin stan- dard. The purpose is to indicate that added endo- toxin is appropriately detected and that the sample does not interfere with the detection of endotoxin. From the results of the Preliminary Testing, a product dilution and possibly product treatment is selected for the Test for Interfering Factors (see below). The endotoxin limit for the product must be detectable at the dilution selected. Test for Interfering Factors (Validation) The Test for Interfering Factors is performed to validate the test conditions and dilution for the particular sample type. It is accomplished by demon- strating that endotoxin added to the sample in PPCs can be readily detected within required limits. Routine Testing Routine testing is conducted using the sample method preparation and conditions for the Test for Interfering Factors and includes a parallel PPC to check for interference. Tests also include negative controls and appropriate standards. Multiple number of units per lot of finished product should be tested, usually sampled from the beginning, the middle and the end of the production run. For medical devices, aqueous extracts of up to ten units are tested, usually after pooling.

• Reagents of differing sensitivity are available: 0.25, 0.125, 0.06 and 0.03 EU/mL

• May be less sensitive to interference than other methods

• Is the referee method as per BET chapters in the United States, European and Japanese Pharmacopeia Overview of Testing Procedures The following section summarizes the procedures/steps to be taken to perform routine product release testing of a sample in a regulated environment. In an unregu- lated environment, or when testing for informational purposes only, follow the procedures described under Preliminary Testing.

Qualification of Reagent, Technician and Laboratory

The reagent must be tested to ensure that it is per- forming to specification. Technicians must be qualified to perform the test and the absence of significant day to day or inter-technician variability in the laboratory should be documented. This requires testing using endotoxin standards only, not samples. Preliminary Testing Preliminary Testing is not a regulatory requirement, but is an important step to develop a set of conditions for the test method that can be used in the Test for Interfering Factors to demonstrate the absence of in- terference. During Preliminary Testing samples should be characterized for endotoxin contamination and/

SUPPORT SERVICES

ACC offers its customers extensive technical support. Our Technical Service department is staffed with experienced professionals who provide customer assis- tance for the full range of ACC products and services. Technical support is available by telephone, email, and in person, through workshops, on-site training, or on-site consultation. Customers who have questions about individual products, test methods, instrumenta- tion, and/or software are invited to call our staff. Software Validation Protocols ACC offers Validation Protocols that provide the end user with a comprehensive set of integrated documents to guide them through the system valida- tion process. The protocol files allow users to edit the documents to meet their company’s specific validation requirements. Reagent Transfer Protocol The Reagent Transfer Protocol document (RTP) is used to validate the change from another manufacturer to ACC BET reagents. If changing BET reagent man- ufacturers, ACC offers assistance with guidance and instructions for using the Validation Protocol. This can be used as verification of the validation process. The Reagent Transfer Protocol is designed to assist users in completing validation of their switch from the current BET reagent to an ACC product. During this process, if the user requires any assistance you will be able to obtain help and advice through the Technical Services department of ACC. Expertise and Resources Assistance with selecting a test method or reagent sensitivity is always available from our Technical Service Department, and representatives in the field. Our staff can help with Preliminary Testing, Testing for Interfering Factors or Routine Testing. The LAL Update ® , our newsletter, includes useful technical articles and is available on our website. Our Contract Test Service (see page 33) team regularly performs Preliminary Testing and method development and can provide results using all test methods. Regardless of which method is selected, you can always be assured of the full support of Associates of Cape Cod, Inc.

For details on endotoxin testing in the United States, users should consult the current revision of the United States Pharmacopia (USP), chapter <85>, “Bacterial Endo- toxins Test”. For those testing outside the US you should consult your local regulatory requirements for the BET. On-Site Consulting Services ACC staff is available to visit client sites to assist investigations and troubleshooting. These visits often address Bacterial Endotoxin Testing (BET) procedures, in addition to identifying sources of contamination in test laboratories and manufacturing processes. #CSOS01 On-site Consulting Services (per day) #SCOS01 On-site Service Call (per day)

Bacterial Endotoxin Testing (BET) Workshop Associates of Cape Cod, Inc. offers training courses on all aspects of bacterial endotoxin testing. Courses an be conveniently conducted on-site or at our facilities in East Falmouth, MA; Liverpool, UK; or Mörfelden-Walldorf, Germany. Customized On-Site Workshops ACC can customize a workshop for you and your staff and conduct it at your facility or ours. Instructors work with you to create a training program tailored to your specific requirements. #WKSP01 1 Day BET Workshop (per workshop, up to 5 attendees) #WKSP02 2 Day BET Workshop (per workshop, up to 5 attendees) #WKSP03 3 Day BET Workshop (per workshop, up to 5 attendees) OSCP-01 1 Day On-site Compounding Pharmacy Training We offer a comprehensive workshop that covers meth- odology, background and in-depth courses, as well as hands-on laboratory experience. A complete schedule can be found on our website, www.acciusa.com. Methodology Background This course is designed to introduce BET methodol- ogies to technicians and managers who are new to endotoxin testing. Topics include: • Endotoxins—What they are, where they come from, and why they are important • BET—An overview of the BET/endotoxin reaction, with emphasis on sources of interference • Detailed instruction of the test methods, including a discussion of laboratory set-up, materials, and aseptic techniques • Sample handling and preparation • Practical approaches to sample characterization and overcoming interference • Technician and laboratory certification and validation of the BET

Hands-On Laboratory The laboratory courses for kinetic and gel-clot methods are designed to give the attendee hands-on experience conducting endotoxin tests. Participants perform tests and learn to read and interpret results. Familiarity with general laboratory techniques (especially pipetting) is essential. In-Depth Topics This course provides the experienced technician with a more detailed understanding of how a BET program can be applied to quality control. Topics include: • Techniques for testing non-aqueous or highly interfering substances • (1 g 3)- b - D -Glucan: contamination, recognition and investigation • Medical device extraction and validation of extraction protocols

• Regulatory considerations Course Schedule and Fees

For course dates and fees, please contact your local ACC representative or check our website at www.acciusa.com. The Bacterial Endotoxin Testing Workshop schedule can be accessed from the BET Products section or from the Calendar section of the ACC website. To receive additional information or to register for a course, contact the appropriate office below. United States: Tel: (800) 848–3248 techservice@acciusa.com United Kingdom: Tel: (44) 151–547–7444 info@acciuk.co.uk Europe: Tel: (49) 61 05–96 10 0 service@acciusa.de

BEST QC Microbiology Training Bioburden, Endotoxin, Sterility Testing Innovative educational programs designed specifically for you.

BEST programs focus on three critical in-process and product release quality control tests:

• Bioburden • Endotoxin • Sterility Testing

BEST is a 3 day innovative educational program designed specifically for you and your laboratory staff. This program will focus on both in-process and product release quality control, including Bioburden, Endotoxin and Sterility Testing. It will provide an overview of relevant methods in each area, however basic technical laboratory skills are assumed as a prerequisite for participation. The course will consist of class presentations and demonstrations of laboratory applications. This program is only available in the US. Program Material Outline

DAY 1 Bioburden Testing Dependable Tests Based on Membrane Filtration

DAY 2 Endotoxin Testing BET Methodology and Background The Bacterial Endotoxin Test (BET) is used for the detection and quantitation of endotoxins from Gram-negative bacteria. Reagents are primarily used to test for endotoxins in injectable pharmaceuticals, biological products, and medical devices. They are also used in renal dialysis centers and a wide range of other applications. During the second day of training, you will learn:

DAY 3 Sterility Testing A Complete Solution for Reliable Results Sterility Testing is considered the most essential QC Microbiological test for releasing sterile final product. This test is heavily regulated and harmonized across most of the globe. During the third day of training, you will learn about:

Bioburden testing is critical for monitoring water quality and raw materials and for ensuring that manufacturing processes remain in microbiological control. During the first day of the training, you will learn about:

• Advantages and limitations of membrane filtration

• The history of Sterility Testing

• The global harmonized regulations overview • Environmental Monitoring requirements for Sterility Testing

• How to choose the right membrane for your application • The regulations governing Bioburden testing • How to develop a sampling plan for Bioburden testing

• What are endotoxin and BET reagents

• The regulations governing bacterial endotoxin testing

• Deep Dive into USP <71>

• The methodology for bacterial endotoxin tetsing

• Advantages and Limitations of Direct Inoculation Sterility Testing • Advantages and Limitations of Open Funnel Sterility Testing • Advantages and Limitations of Closed System Sterility Testing • Overview of Sterility Testing Media and Rinse Fluids

• How to qualify a chosen BET method

• How to qualify and validate a method

• How to validate samples and how to test them routinely

• How to set alert and action limits

• How to interpret Bioburden test results

• How to analyze and interpret data

• How to troubleshoot membrane filtration issues

• How to address sample interference

• Hands-on session - Pyros Kinetix ® Flex tube reader and Pyros ® eXpress software

• Rapid methods for Bioburden testing

• Most Common Sterility Questions

• Hands-on session using a manifold and Milliflex Plus Pump

• Learn about sustainable recombinant reagents

• Hands-on session - Steritest Equinox

For more information go online to: www.acciusa.com/products-and-services/bet-training-and-support/best-workshop

KINETIC ENDOTOXIN DETECTION SYSTEMS

Our Test, Your Cure...

Ensuring A Healthy World

Your Endotoxin Experts! Associates of Cape Cod, Inc. offers diverse options for conducting endotoxin testing.

Another First!

rCR, Brought To You By The BET Pioneers Associates of Cape Cod, Inc.

A History Of Firsts! First To Introduce An Animal Free, Recombinant LAL Reagent First Large Scale IVF Program To Introduce Horseshoe Crabs Into The Wild First To Receive FDA License For All Three BET Test Methods First BET Company Licensed By FDA

KINETIC CHROMOGENIC METHOD RECOMBINANT REAGENT

PyroSmart NextGen ® Recombinant Cascade Reagent (rCR)

Sample to Lysate Ratio PyroSmart NextGen ® is used at with an economical vol- ume of 50 µL of reagent per well yielding 50 tests/vial:

• Microplate reader: 1:1 ratio using 50 µL of test sample : 50 µL of reagent

Test Performance The PyroSmart NextGen ® reaction mixture is incubated at 37±1°C and read in a microplate reader equipped with a 405–410 nm filter. The time of incubation is dependent on the lowest standard concentration in the standard curve, with 0.005 EU/mL achievable in 2500 seconds in a microplate reader. Software is used to construct the standard curve and calculate the endotoxin concentrations. Product Stability PyroSmart NextGen ® is a lyophilized product with an excellent shelf life of 3 years from the date of manufacture. Product Reconstitution PyroSmart NextGen ® is provided as co-lyophilized with the chromogenic substrate and as such it is ready-to-use following a simple reconstitution (using 2.8 mL of the supplied reconstitution buffer). Product Packaging PyroSmart NextGen ® reagent is provided as a pack of 2 vials of reagent and 2 vials of reconstitution buffer. This is sufficient for a total of 110 wells (55 wells per vial).

PyroSmart NextGen ® recombinant Cascade Reagent (rCR) marks the introduction of a new sustainable recombinant LAL reagent technology for Bacterial Endotoxin Testing (BET). Utilizing the same LAL cascade as traditional LAL reagents, while eliminating the potential for (1 g 3)- b - D -Glucans cross reactivity, PyroSmart NextGen ® delivers all of the quality and consistency of results you have come to expect from ACC LAL reagents. PyroSmart NextGen ® can be used for a wide variety of endotoxin tests, ranging from standard water testing to samples requiring high sensitivity, such as intrathecal products and those requiring high dilutions to overcome interference. Product Sensitivity The sensitivity for recombinant chromogenic assays is determined by the lowest standard concentration on the standard curve used for the assay. The maximum sensitivity of PyroSmart NextGen ® is 0.005 EU/mL when run in an incubating microplate reader (or 0.001 EU/mL when run in Pyros Kinetix ® Flex tube reader).

Multi-Test 2.8 mL/vial (approx. 50 tests/vial) #PNG050-2 Each kit contains

2 x PyroSmart NextGen ® Reagent 2 x Reconstitution Buffer

KINETIC CHROMOGENIC METHOD RECOMBINANT REAGENT

PyroSmart NextGen ® is a sustainable recombinant Cascade Reagent (rCR) that delivers the same reliable results as your conventional LAL reagent and offers these additional advantages: • No Animal Content – Horseshoe Crab Blood Free • Same Cascade • No Cross Reactivity With 1,3-b-D-glucans • Same Instrument • Same Preparation Steps • Meets Your Sustainability Objectives Keep Your Method. Make An Impact.

ACC’s PyroSmart NextGen ® uses the same cascade as traditional LAL reagents by manufacturing the Factors responsible for the cascade using recombinant processes. As a result, our new recombinant reagent’s mechanism of action will deliver results consistent with traditional LAL reagents. It offers the added advantage of elim- inating 1,3-b-D-glucans cross reactivity from the LAL cascade, since there is no Factor G in the final reagent. ACC developed Pyrosmart NextGen ® to provide a sustainable alternative to traditional naturally sourced LAL reagents, while maintaining your lab procedures, methods, instrumentation and most importantly your results.

The Importance of Mechanism of Action Recombinant Cascade Reagent (rCR)

Recombinant Cascade Reagent (rCR)

Recombinant Factor C Reagent (rFC)

Traditional LAL Reagent

Endotoxin

rFC

Glucan

Glucan No Factor G

rFB

Fluorogenic Substrate

Product Fluorescence

r Proclotting Enzyme

r Clotting Enzyme

Proclotting Enzyme

Clotting Enzyme

Coagulogen Turbidimetric

Coagulin Turbidity Product Yellow Color

Chromogenic

Product Yellow Color

Chromogenic Substrate

Chromogenic

Chromogenic Substrate

Traditional LAL reagent In the presence of endotoxin, Factor C becomes an activated moiety which in turn activates Factor B and Proclotting Enzyme; ultimately result- ing in the proteolytic cleavage of a substrate (either coagulogen in gel clot and turbidimetric assays or a colorless chromogenic substrate in chromogenic assays). The cascade mechanism thus amplifies the response of Factor C and leads to an excep- tional sensitivity for this biological assay, with kinetic output being preferable. In the pres- ence of 1,3- b - D -glucans, Factor G becomes an activated moiety which also activates Proclotting Enzyme and thus resulting in the same signal as that triggered by endotoxins through Factor C. This has been often observed as glucan-derived enhancement or false positive results.

Recombinant Cascade Reagent (rCR) As with naturally sourced LAL reagents, in the presence of endotoxin, recombinant Factor C becomes an activated moiety which in turn activates recombinant Factor B and recombinant Proclotting Enzyme; ultimately resulting in the proteolytic cleavage of a colorless chromogenic substrate formulated with PyroSmart NextGen ® . By relying on the same cascade mechanism, the response of recombinant Factor C is amplified the same way as by LAL reagents and thus the same sensitivity is achieved using this kinetic assay. Due to absence of Factor G, PyroSmart NextGen ® will not react with any 1,3- b - D -glucans and therefore will prevent glucan-derived enhancement and false positive results.

Recombinant Factor C (rFC) – Competition

Launched almost two decades ago, rFC reagents rely only on a recombinant form of Factor C. Due to the absence of the cascade as the amplification mech- anism, rFC reagents are paired with a fluorescence method instead. However, this constitutes a dif- ferent measured entity, different instrumentation, and different preparation steps with a limited output (endpoint assay only). Therefore the uptake and implementation of this method has been rather limited.

The Benefits Are Clear

ACC’s PyroSmart NextGen ®

LAL Reagent

Conventional LAL Reagent

Competitor (rFC) Reagent

(rCR) Reagent

Comparison Table

2021

Year Technology Introduced

1977

2003

O No. Endpoint only

Kinetic Assay

Kinetic

P Kinetic

O No. rFC requires three reagents in a 1:4:5 ratio and a 10 min. pre-incubation step

Assay Setup

Single step

P Single step

reconstitution

O No. Fluorescent reader required

P Yes. Incubating plate or tube reader at 405 nm

Same Standard Plate Reader

Incubating plate or tube reader at 405 nm

O No. Based on Carcinoscorpius

P Yes. rCR is recombinant LAL

Derived From

LAL

Limulus Amebocyte Lysate (LAL)

or Tachypleus Amebocyte Lysate (CAL/TAL)

O No

P Yes

Multi-step Cascade Pathway

Yes

Endotoxin Specific

P Endotoxin Specific

Sustainable Reagent (animal free)

P Horseshoe Crab Blood Free

Converting to PyroSmart NextGen ® Is Easy Switching to this sustainable alternative is easy because PyroSmart NextGen ® follows the same cascade pathway as traditional reagents. But don’t take our word for it – evaluate PyroSmart NextGen ® yourself on your existing absorbance readers. Follow our user-friendly Evaluation Protocol to determine if PyroSmart NextGen ® works in your laboratory and on your samples. Our experts will assist you every step of the way. If interested, simply contact an ACC Account Manager or Distributor today.

Keep Your Method. Make An Impact. • Same Instrument • Same Preparation Steps • Same Method The future of sustainable LAL testing has arrived with ACC’s PyroSmart NextGen ® recombinant LAL reagent. The new testing technology that delivers the best of traditional methods, combined with the benefits of sustainable recombinant Cascade Reagents (rCR). The future of LAL testing is bright!

Keep Your Method. Make An Impact.

KINETIC CHROMOGENIC METHOD REAGENTS

Performing the Test The Pyrochrome ® sample mixture is incubated in an optical reader at 37±1°C and read at a wavelength of 405 nm. No pre-incubation is required and results can be available within 1 hour. However, time to results is dependent on the required assay sensitivity. Software will analyze the data to provide endotoxin results. Reconstitution Pyrochrome ® lysate is reconstituted with an optimized Pyrochrome ® reconstitution buffer (C1500-5). Pyro- chrome ® can also be reconstituted with Glucashield ® buffer (CG1500-5), a (1 g 3)- b - D -Glucan inhibiting buffer, to render the assay endotoxin specific. Stability Once reconstituted, Pyrochrome ® is stable for 8 hrs. when stored at 2–8ºC. Packaging Pyrochrome ® is offered with a choice of reconstitu- tion buffer and is recommended for use with the 10 ng/vial Control Standard Endotoxin (CSE, EC010-5). Certificates of Analysis, specific to the Pyrochrome ® and CSE lot, can be obtained from ACC or online at www.acciusa.com. Pyrochrome ® Chromogenic Test Kit 3.2 mL/vial (approx. 60 tests/vial) Each kit contains Pyrochrome ® and a buffer #C1500-5 Pyrochrome ® with Reconstitution Buffer 5 pack (300 tests) #C1500-25 Pyrochrome ® with Reconstitution Buffer 25 pack (1500 tests) #CG1500-5 Pyrochrome ® with Glucashield ® Buffer 5 pack (300 tests) #CG1500-25 Pyrochrome ® with Glucashield ® Buffer 25 pack (1500 tests)

The BET reagent is formulated with a synthetic substrate which produces a chromophore when cleaved by endotoxin activated enzyme. The test is read in a tube reader or an incubating microplate reader. Pyrochrome ® Reagent Chromogenic Endotoxin Testing

Pyrochrome ® can be used for a wide variety of sample types, ranging from standard water testing to samples requiring high sensitivity, such as intra- thecal products and those requiring high dilutions to overcome interference. Sensitivity The maximum sensitivity of Pyrochrome ® is 0.001 EU/mL when run in Pyros Kinetix ® Flex tube reader or incubating microplate reader with Glucashield ® Buffer. Sample to BET Ratio In the Pyros Kinetix ® Flex tube reader, Pyrochrome ® can be used at an economical ratio of 4:1 using 50 μL of reagent per well or at 1:1 using 100 μL/well. In a microplate reader, the reagent is used at a ratio of 1:1 and a volume of 50 μL/well (60 tests/vial) or 100 μL/well (30 tests/vial).

Chromo-LAL

Stability Once reconstituted, Chromo-LAL is stable for 24 hours if stored at 2–8°C. Chromo-LAL may be frozen once and will retain activity for 2 weeks if stored at or below -20°C. Packaging Each vial contains reagent for approximately 30 tests. It is recommended for use with 0.5 μg/vial Control Standard Endotoxin (CSE, E0005-1). Certificates of Analysis, specific to the Chromo-LAL and CSE lot, can be obtained from ACC or online at www.acciusa.com.

Chromo-LAL #C0031-5 3.2 mL/Vial 5 Pack (150 tests)

Chromo-LAL is optimized for the kinetic chromogenic BET test method in microplate readers. Chromo-LAL is a buffered, stable and robust lysate, suitable for quan- titative testing of a wide range of samples. Sensitivity The sensitivity for chromogenic assays is determined by the lowest standard concentration on the standard curve used for the assay. The maximum sensitivity of Chromo-LAL is 0.005 EU/mL. Sample to BET Ratio Reconstituted Chromo-LAL reagent is used at a ratio of 1:1 and a volume of 100 μL/well (30 tests/vial). Performing the Test The Chromo-LAL/sample mixture is incubated at 37±1°C and read in a microplate reader. Software is used to construct the standard curve and calculate the endotoxin concentrations. Reconstitution Chromo-LAL lysate is reconstituted with BET Reagent Water (LRW). It can also be reconstituted with Gluca- shield ® buffer, a (1 g 3)- b - D -Glucan inhibiting buffer, to render the assay endotoxin specific.

KINETIC TURBIDIMETRIC METHOD REAGENT

The optical density (turbidity) increase that accompa- nies the clotting reaction is read in the Pyros Kinetix ® Flex tube reader or in an incubating microplate reader. Pyrotell ® -T Reagent Turbidimetric Endotoxin Testing

to lysate ratio of 1:1 or 4:1 and volume of 100 μL/well (48 tests/vial) or 50 μL/well (96 tests/vial), respectively. Performing the Test The Pyrotell ® -T sample mixture is incubated in an optical reader at 37±1°C and read at a desired wave- length depending on the instrumentation and user choice. The time of incubation is dependent on the lowest standard concentration in the standard curve. Software is used to analyze the standard curve and calculate the endotoxin concentrations. Reconstitution Pyrotell ® -T may be reconstituted with 5 mL of LAL Reagent Water (LRW), Pyrosol ® buffer, or Glucashield ® buffer, depending on the demands of the sample be- ing tested. Pyrosol ® buffer provides improved kinetics and extra pH buffering capacity. Glucashield ® buffer, a (1 g 3)- b - D -Glucan inhibiting buffer, is used to render the assay endotoxin specific. Stability Once reconstituted, Pyrotell ® -T is stable for 24 hours, if stored at 2–8°C. Pyrotell ® -T may be frozen once and will retain activity for as long as 3 months if stored at or below -20°C. Packaging Pyrotell ® -T is available in multi-test vials. Each vial contains reagent for approximately 96 tests (when used with the Pyros Kinetix ® Flex tube read- er and 4:1 sample to BET ratio) or 48 tests (when used with 1:1 ratio and/ or in a microplate read- er). It is recommended for use with the 0.5 μg/ vial Control Standard Endotoxin (CSE, E0005-1). Certifi- cates of Analysis, specific to the Pyrotell ® -T and CSE lot, can be obtained from ACC or online.

Pyrotell ® -T formulation is a versatile and cost effective solution for the determination of endotoxin. It can be used with the Pyros Kinetix ® Flex tube reader and incubating mi- croplate readers. When used with the Pyros Kinetix ® Flex tube reader, Pyrotell ® -T is a highly economic, flexible and sensitive BET assay. Turbidimetric reagent It can be used for a wide variety of tests, ranging from water testing to samples requiring high sensitivity, such as intrathecal products and those requiring high dilutions to overcome interference. Sensitivity When used in a Pyros Kinetix ® Flex tube reader the maximum sensitivity is 0.001 EU/mL. The unique formulation of Pyrotell ® -T allows a wide selection of standard curves to be used, giving the user flexibility, speed and ease in performing assays. Sample to BET Ratio The ratio of sample to BET is determined by personal preference and sample chemistry (interference patterns). Reconstituted Pyrotell ® -T reagent is used at a sample

Pyrotell ® -T Multi-Test 5 mL/vial (approx. 50 tests/vial) #T0051-5 5 pack (250 tests) #T0051-25 25 pack (1250 tests)

PYROS KINETIX ® FLEX CHROMOGENIC AND TURBIDIMETRIC TUBE READER

• Flexible Testing: Variable volumes and ratios can be utilized for increased efficiency, ability to maximize product Maximum Valid Dilution (MVD)

• Reduced Lysate Usage: As little as 50 µL per test

• Increased Efficiency: Unlike plate readers, end-users have the ability to add samples to an existing assay in order to efficiently utilize all wells. Software provides individual sample results while assay continues to run.

Pyros Kinetix ® Flex Designed with flexibility and efficiency in mind, the PK Flex provides flexible options for conducting endotoxin testing. The Pyros Kinetix ® Flex instrument and Pyros ® eXpress 21 CFR Part 11 compliant Software provide a complete system for efficient and accurate endotoxin testing. Features & Benefits • Capable of running either Chromogenic and Turbidi- metric testing in the same instrument • Broad Sensitivity Range: No other system is more sensitive; As high as 0.001 EU/mL with either our Pyrochrome ® chromogenic reagent or our Pyrotell ® -T turbidimetric reagent

• Uses low cost depyrogenated glass disposable accessories.

• Solid State Design: Low instrument maintenance

System Specifications Capacity: 32, 64 or 96 reaction tubes

Power Requirement: 100 to 240 VAC @ 50 / 60 Hz

Light Source: LED

Dimensions & Weight: #PKF32 - 9.25” x 10” x 3.125” / 6 lbs 15 oz #PKF64 - 9.25” x 14” x 3.125” / 11 lbs 5 oz #PKF96 - 9.25” x 18” x 3.125” / 15 lbs 14.5 oz

Precise Temperature Control: Incubator temperature is held to 37°C ± 0.5°C

Two Wavelength Settings (405 nm and 660 nm)

Warranties, Parts and Service For details on extended warranties, repairs and recalibrations, contact your supplying US, UK, German office or your local representative/distributor.

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